Eukaryotic chromatin structure and gene expression are regulated by histone acetylation by histone acetyltransferase (HAT), and deacetylation by histone deacetylase (HDAC). HDAC inhibitors are already known to induce cancer cell differentiation and apoptosis, and are expected to be useful as antitumor agents (Non-Patent Documents 1-3). In fact, clinical studies have begun in the United States for some HDAC inhibitors (on-Patent Documents 4 and 5) that are effective as antitumor agents in animal experiments.
Tricostatin A (TSA) is well known as a specific HDAC inhibitor (Non-Patent Document 6). In fact, TSA has been known to induce differentiation to leukemia cells, neuronal cells, breast cancer cells, and the like (Non-Patent Documents 7-14). Furthermore, the TSA activities of differentiation induction and apoptosis induction are known to synergistically increase when used in combination with drugs that activate gene expression by mechanisms different from HDAC inhibitors. For example, cancer cell differentiation can be promoted by using HDAC inhibitors in combination with retinoic acids, which activate retinoic acid receptors that serve as nuclear receptors, thereby inducing gene expression relevant to differentiation (Non-Patent Documents 9, 13, 15, and 16). 5-azadeoxycytidine inhibits DNA methylation to reduce expression of tumor suppressor genes in many cancer cells. TSA used in combination with 5-azadeoxycytidine promotes cancer cell apoptosis and restoration of tumor-suppressing gene expression (Non-Patent Documents 17-21).
HDAC inhibitors are expected to act not only as antitumor agents but also as cancer preventives. TSA, SAHA, and the like significantly suppressed the occurrence of breast cancer induced in animal models. Also, investigations carried out using valproic acids indicated that HDAC inhibitors suppress metastasis (Non-Patent Document 14).
In addition to applications as antitumor agents, HDAC inhibitors have other applications, including as therapeutic and ameliorative agents for autoimmune diseases, neurodegenerative diseases such as polyglutamine disease (Non-Patent Documents 22 and 23), skin diseases, and infectious diseases (Non-patent Document 24). HDAC inhibitors are also used to improve efficiency of vector transfer for gene therapy (Non-Patent Document 25), and enhance expression of transferred genes (Non-Patent Document 26). HDAC inhibitors may also have inhibitory effects against angiogenesis (Non-Patent Document 27 and 28).
There are 10 or more subtypes of HDAC, and recently, certain HDAC subtypes have been found to be closely related to cancer. For example, for tumor suppressor gene p53, which plays a very important role in inhibiting cancer development, to express its faction, acetylation of p53 itself is important (Non-Patent Document 29) and HDAC1 and HDAC2 have been found to be involved in inhibiting this function (Non-Patent Document 30). It has also been demonstrated that HDAC4 and the like are recruited via nuclear corepressors by oncogenes, such as PML-RAR and PLZF-RAR, proteins involved in development of acute promyelocytic leukemia (APL) and Bcl-6, involved in the development of lymphoma, and lead to cancer development by inhibiting the expression of a group of genes required for normal differentiation (Non-Patent Documents 31-34). In addition, among tissue-specifically expressed HDAC subtypes, some are known to play important roles in the development and differentiation of normal tissues (Non-Patent Documents 35 and 36).
HDAC6 is an enzyme which is shuttled between the nucleus and the cytoplasm by nucleo-cytoplasmic transport, and which normally locates in the cytoplasm (Non-Patent Document 37). HDAC6 is highly expressed in the testes, and is presumed to relate to the differentiation of normal tissues. Furthermore, HDAC6 is known to be associated with microtubule deacetylation, and to control microtubule stability (Non-Patent Document 38). HDAC6 is also a deacetylation enzyme bonded to a microtubule and affecting cell mobility (Non-Patent Document 39). Accordingly, HDAC6 inhibitors may find utility as metastasis-suppressing agents. TSA inhibits each HDAC subtype to about the same degree. However, HDAC6 cannot be inhibited by trapoxins comprising cyclic tetrapeptide structure and epoxyketone as active groups (Non-Patent Document 40). Based on the information on the three-dimensional structure of the enzyme, trapoxins are presumed to exert poor binding properties to HDAC6 due to the structure of its cyclic tetrapeptide moiety that interacts with the weakly conserved outward surface of the enzyme active center. Accordingly, altering the cyclic tetrapeptide portion may result in inhibitors that are selective for a variety of HDAC.
TSA shows inhibitory activity due to the coordination of its hydroxamic acid group with zinc in the HDAC active pocket (Non-Patent Document 41). Examples of known HDAC inhibitors comprising hydroxamic acid include Oxamflatin (Non-Patent Document 42) and CHAP (Non-Patent Documents 40 and 43). However, TSA is unstable in blood and has a strong hydroxamic acid chelating function. It also chelates with other essential metal ions. Therefore, HDAC inhibitors comprising hydroxamic acid have not actually been used as antitumor agents to date. Meanwhile, thiol groups produced by the reduction of FK228 disulfide bonds have recently been shown to serve as active groups to be coordinated with zinc in the HDAC active pocket, thereby inhibiting HDAC. Thus, FK228 is a prodrug that is activated when reduced by cellular reducing activity (Non-Patent Document 44).
Furthermore, a number of HDAC inhibitors comprising cyclic tetrapeptide structures and epoxyketones as active groups have been isolated Tom natural environments. On the basis of such findings, the cyclic tetrapeptide structure is suggested to be useful in enzyme identification (Yoshida, et al., 1995, supra); however, from various viewpoints such as stability, existing inhibitors have not advanced to the level of being satisfactorily qualified to be pharmaceutical products. Therefore, production of pharmaceutical agents in which these problematic points have been resolved is strongly anticipated.
Prior art documents relating to the invention of the present application are listed below.    [Non-Patent Document 1] Marks, P. A., Richon, V. M., and Rifkind, R. A. (2000) Histone deacetylase inhibitors: Inducers of differentiation or apoptosis of transformed cells. J. Natl. Cancer Inst. 92, 1210-1216    [Non-Patent Document 2] Yoshida, M., Horinouchi, S., and Beppu, T. (1995) Trichostatin A and trapoxin: novel chemical probes for the role of histone acetylation in chromatin structure and function. Bioessays 17, 423-430    [Non-Patent Document 3] Bernhard, D., Loffler, M., Hartmann, B. L., Yoshida, M., Kofler, R., and Csordas, A. (1999) Interaction between dexamethasone and butyrate in apoptosis induction: non-additive in thymocytes and synergistic in a T cell-derived leukemia cell line. Cell Death Diff. 6, 609-617    [Non-Patent Document 4] Nakajima, H., Kim, Y. B., Terano, H., Yoshida, M., and Horinouchi, S. (1998). 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